Amyloids are filamentous polymers of aberrantly folded proteins distinguished by cross-beta structure. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, Type 2 diabetes, and rheumatoid arthritis. Amyloids are distinguished into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focussing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model;investigating fibril polymorphism;and relating amyloids to native conformations. In FY11 we focussed on three areas:&#8232;&#8232; (1) Systematic analysis of beta-arcade motifs in amyloid fibril models and beta-solenoid protein structures. Unlike the diverse folds of native proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and in having cross-beta structures. Despite the difficulties attendant upon obtaining high resolution experimentally determined fibril structures, increasingly credible models are being derived by integrating data from multiple techniques. Most current models of disease-related amyloids invoke beta-arcades, columnar structures produced by in-register stacking of beta-arches. A beta-arch is a strand-turn-strand motif in which the two beta-strands interact via their side-chains, not via the polypeptide backbone as in a conventional beta;-hairpin. Crystal structures of beta-solenoids, a class of proteins with amyloid-like properties, offer insight into the beta-arc turns found in arches. General thermodynamic considerations suggest that complexes of two or more beta-arches may nucleate amyloid fibrillogenesis.&#8232;In FY11, we published a review of beta-arcades (1). (2) Visualization of Ure3 prion filaments in infected yeast cells. Wild-type Ure2p is a soluble dimeric protein that contributes to regulating nitrogen catabolism in S. cerevisiae. In its prion form, Ure2p aggregates and loses its activity. Our earlier work on thin section electron microscopy of infected cells over-expressing Ure2p showed that the aggregates have a filamentous substructure and, by immuno-gold labelling, that the filaments contain Ure2p. In vitro, Ure2p assembles into filaments with an amyloid fibril backbone surrounded by globular domains. In FY11, we extended this line of investigation with the aims of making a closer comparison between in vitro-assembled and in vivo=assemble filaments and accounting for the total cellular complement of Ure2p. To this end, we used electron tomography. In specimens whose preservation was optimized by freeze substitution, the filaments are seen to be non-hollow and 20 nm in diameter. In vitro-assembled filaments prepared in identical fashion are also non-hollow and of essentially the same diameter. In aggregates, the filaments are randomly oriented with occasional crossing points. We did not observe any connection of aggregates to other cytoplasmic components;ribosomes, otherwise abundant in the cytoplasm, are excluded. By comparing the amount of Ure2p per cell, determined biochemically, with the amount in filaments, quantitated from tomograms, we conclude that most if not all Ure2p is present in the aggregates. These observations are being prepared for publication. (3) Disposition and role of the highly charged middle domain (M-domain) of the Sup35p prion protein. In yeast cells infected with the PSI+ prion, the protein Sup35p forms aggregates and its activity in translation termination is down-regulated but not eliminated. Sup35p has an N-terminal prion domain;a highly charged M-domain of about 125 residues;and a functional C-terminal domain. By negative staining, cryo-EM, and scanning transmission EM (STEM), in vitro-assembled filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse cloud of C-domains, giving a full diameter of 65nm. In diameter (8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield 1 subunit per 0.47 nm for N-fibrils, NM-fibrils, and Sup35p filaments, further supporting the amyloid fibril backbone model. The 30nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations. The extended M-domain conformations offer an explanation for residual Sup35p activity in infected cells, whereby the C-domains remain free enough to be capable of some interaction with ribosomes. In FY11, this project was completed and published (2). Current research is focussed primarily on the production of well ordered filaments of Ure2p, intended for high resolution cryo-EM analyses of the backbone structure.